The vertebrate genome contains a predicted 20,000+ genes, many of unknown biological role(s). In addition, a large fraction of these molecules have distinct functions in diverse processes. The functional annotation of the vertebrate genome is the overarching goal of this long-standing research program. For example, even a decade after the completion of the human genome effort, the diversity of genes undergoing active research has not substantially changed. We developed a collection of gene-break transposon (GBT) alleles for use in phenotypic annotation of the vertebrate genome using the preeminent non-mammalian model organism, the zebrafish (Danio rerio). Why emphasize the proteome? Genomic approaches have robustly characterized the nuclear genome and transcriptome. However, the genomic assessment of the full complexity of the proteome in a dynamic context and in vivo is still largely unknown. The zebrafish provides an opportunity to perform a comprehensive analysis of the entire vertebrate proteome. We will use our ongoing 700+ GBT collection and new, targeted TALEN/CRISPR knockout alleles for assessment of the vertebrate phenome. In this competitive renewal, we will focus on the annotation of biological function to identify new players in development as well as a set of clinically relevant biological processes. We will accomplish this goal through the following specific aims: Specific Aim I. We will annotate nuclearly encoded mitochondrial genes during vertebrate development using zebrafish. Specific Aim II. We will annotate novel genes with expression and function in the embryonic and adult skin and will analyze their contribution to organ homeostasis and regenerative potential. Specific Aim III: We will identify cardiac mutants for annotation of gene modifiers of adult cardiomyopathy. Specific Aim IV. We will identify digestive organ mutations to characterize a variety of potentially disease-causing mutant lines for alterations in lipid uptake, metabolism, transport and storage. We selected 1) Mitochondrial biology due to the complex role of this organelle in normal biology and disease and the unexpected finding of diverse phenotypes from the first GBT alleles in nuclearly encoded mitochondrial genes embryonic 2) Integument development and function due to the strong impact of skin disease on one in three Americans 3) Cardiac biology and function due to the critical role of heart disease as the leading cause of death in the US and 4) Lipid biology and digestive disease physiology due to their critical role(s) in development, heart disease and obesity. Together, this program will provide new insights into the genetic basis of new vertebrate developmental processes and physiological systems critical for health and that when altered contribute to disease.